Cardiovascular disease is the leading cause of death for men and women regardless of ethnicity and up to 25% of the population live with some form of chronic cardiovascular disease. Reduced capacity of the endothelium to produce nitric oxide (NO), via the enzyme endothelial nitric oxide synthase (eNOS), is a hallmark of many cardiovascular diseases yet the mechanisms regulating eNOS activity are not fully understood. In addition to transcriptional control, eNOS is regulated by post-translational mechanisms including calcium, phosphorylation, protein-protein interactions and subcellular localization. Within endothelial cells, both cultured and in blood vessels, eNOS is localized to the plasma membrane and the perinuclear Golgi complex. The relative proportion of eNOS in both locations is variable and a recently discovered eNOS binding protein, NOSIP, regulates the amount of eNOS within these domains. However, the individual function of eNOS at the Golgi or plasma membrane is not yet known. In preliminary results we have shown that by targeting eNOS exclusively to the Golgi or plasma membrane, intracellular location can directly influence both the amount of NO released and the mechanisms leading to eNOS activation. Plasma membrane eNOS has elevated basal activity, is calcium-sensitive and is constitutively phosphorylated on serine 1179. In contrast, Golgi eNOS displays lower levels of calcium-dependent activity and is highly activated by Akt mediated phosphorylation. Based on these findings, the central hypothesis of this proposal is that agonists utilizing Akt- dependent pathways preferentially activate Golgi eNOS and that calcium-dependent agonists preferentially activate plasma membrane eNOS. To test this hypothesis, 4 specific aims are proposed: The first aim will determine whether agonists utilizing Akt or calcium-dependent mechanisms preferentially activate Golgi or plasma membrane eNOS. The second aim will determine the mechanisms (calcium and phosphorylation) underlying the divergent activities of Golgi and plasma membrane eNOS. The third aim will determine whether the protein NOSIP concentrates eNOS in the Golgi and thus modulates the activity of specific agonists. The fourth aim will determine the importance of eNOS subcetlutar targeting to vascular function in isolated blood vessels.